Changes in the expression and functional activities of Myosin II isoforms in human hyperplastic prostate

Benign prostatic hyperplasia (BPH) is a common disease among aging males with the etiology remaining unclear. We recently found myosin II was abundantly expressed in rat and cultured human prostate cells with permissive roles in the dynamic and static components. This study aimed to explore the expression and functional activities of myosin II isoforms including smooth muscle myosin II (SMM II) and non-muscle myosin (NMM II) in the hyperplastic prostate. Human prostate cell lines and tissues from normal human and BPH patients were used. H&E, Masson’s trichrome, immunohistochemical staining, in vitro organ bath, RT-PCR and Western-blotting were performed. We further created cell models with NMM II isoforms silenced and proliferation, cycle, and apoptosis of prostate cells were determined by CCK-8 assay and flow cytometry. Hyperplastic prostate SM expressed more SM1 and LC 17b isoforms compared to their alternatively spliced counterparts, favoring a slower more tonic-type contraction and greater force generation. For BPH group, blebbistatin (BLEB, a selective myosin II inhibitor), exhibited a stronger effect on relaxing phenylephrine (PE) pre-contracted prostate strips and inhibiting PE induced contraction. Additionally, NMMHC-A and NMMHC-B were upregulated in hyperplastic prostate with no change in NMMHC-C. Knockdown of NMMHC-A or NMMHC-B inhibited prostate cell proliferation and induced apoptosis, with no changes in cell cycle. Our novel data demonstrates that expression and functional activities of myosin II isoforms are altered in human hyperplastic prostate, suggesting a new pathological mechanism for BPH. Thus, the myosin II system may provide potential new therapeutic targets for BPH/lower urinary tract symptoms (LUTS).


Introduction
Benign prostatic hyperplasia (BPH) is one of the most common disease among aging males. Its incidence increases with age and reaches 90% at the age of 85 (1). The pathophysiology of BPH has two components, one is a static component that causes increased prostate volume, and the other is a dynamic component resulting in increased prostatic tone (active and passive tension) and bladder decompensation and overactivity (1). Although aging and androgens play important roles in the development of BPH, its exact etiology remains unclear. Recently, we demonstrated that myosin II was abundantly expressed in the rat prostate where it could regulate prostatic tone and cell proliferation (2). However, the myosin II in human prostate was less well studied. Exploring myosin II in human hyperplastic prostate may provide new insight into the pathophysiological mechanisms and therapeutic targets for BPH.
Myosin II, a motor protein in eukaryotic cells, induces a wide range of biological functions when coupled with actin (3). Myosin II includes skeletal muscle myosin II, cardiac myosin II, smooth muscle (SM) myosin II (SMM II) and non-muscle myosin II (NMM II). The expression of the three muscle myosins is restricted largely to their respective muscle tissue, while NMM II is expressed in most mammalian cells including muscle cells. Our recent study demonstrated that SMM II is a contractile apparatus which mainly regulated prostatic SM tone while NMM II may regulate prostatic cell proliferation (2).
SMM II is a hexameric molecule consisting a pair of myosin heavy chains (MHC) and two pairs of myosin light chains (MLC, both MLC 17
Meanwhile, we also demonstrated that SMM II isoform composition and contractility profile is altered in the castrated rat prostate, which expresses more SM2 and SM-B but less LC 17a , favoring a faster more phasic-type contraction (15). However, SMM II isoform composition and correlating contractility profiles for human prostate remain undefined. Moreover, whether the expression and functional activity of SMM II is altered in hyperplastic prostate is worth exploring.
Similar to the structure of SMM II, NMM II is also a hexameric molecule consisting of three components: a pair of MHCs, a pair of regulatory MLCs that regulate NMM II activity and a pair of essential MLC that stabilize the MHC structure.
There are three NMMHC isoforms (NMMHC-A, NMMHC-B and NMMHC-C), encoded by three different genes (MYH9, MYH10 and MYH14), respectively (16)(17)(18). In contrast to the role of SMM II in mediating prostatic SM tension, NMM II was demonstrated to play important roles in cellular "housekeeping"-type processes, including cell proliferation, adhesion, migration, along with synthetic and secretory functions (3,19). Since NMM II is highly expressed in embryonic tissues and downreguled in mature rats (20), NMM II is also referred to embryonic myosin Ⅱ.
NMMHC-C was highly expressed in mouse cochlea and was crucial for auditive function (26). New glands, which can only be seen in the embryonic period, are often found in human hyperplastic prostate. It therefore has been proposed that the occurrence of BPH is the "reawakening" of the embryonic process involving prostate mesenchyme induced epithelial differentiation (27). Our recent study also identified these three NMM II isoforms in rat prostate tissues and cultured human prostate cells, and we showed that NMM II might play an important role in cell proliferation and BPH development (2). Again, these NMM II isoforms were less well studied in the human hyperplastic prostate.
Currently, with regard to the therapeutic treatment of BPH, 5α-reductase inhibitors (reducing prostate volume) and α-adrenergic blockers (decreasing prostatic SM tone) are the first line agents and are effective per se. However, side effects frequently occurr such as dizziness, asthenia and sexual dysfunction. In addition, approximately 30% of BPH patients still require surgical treatment (28). Therefore, it Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20201283/900934/cs-2020-1283.pdf by guest on 05 January 2021 is necessary to identify new therapeutic targets for BPH. Blebbistatin (BLEB), a small cell permeable selective myosin II inhibitor, was originally discovered as an inhibitor of NMM II (9). Our recent study found that BLEB significantly inhibits proliferation of human epithelial and stromal cells in vitro, and that intra-prostatic injection of BLEB can reduce the volume of the rat prostate (29). In addition, BLEB has been suggested to inhibit SM contraction with near equipotency as for NMM II (30)(31)(32). We also demonstrated that BLEB could relax rat prostate strips in a dose-dependent manner (33). However, the effect of BLEB on human normal and hyperplastic prostate has been less well examined and whether its efficacy is altered in the hyperplastic prostate remains unknown.

Human tissue acquisition.
Human hyperplastic prostate tissues were obtained from ten male patients (mean age, 68.3±3.5 years) needing to undergo radical cystectomy for infiltrating bladder cancer. All samples showed BPH and no tumor infiltration, which were identified by two separate pathologists. Normal prostate tissue was obtained from ten brain-dead men (mean age, 31.7±2.5 years) undergoing donation at the Organ Transplant Center of Zhongnan Hospital, with pathological examination revealing no hyperplasia.
Additionally, aorta, vena cava, corpus cavernosum (CC) and bladder tissues from normal humans were also obtained. All human samples were obtained after the approval of the Hospital Committee for Investigation in Humans and after receiving Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20201283/900934/cs-2020-1283.pdf by guest on 05 January 2021 written informed consent from all patients or their relatives. All human studies were conducted in accordance with the principles of the Declaration of Helsinki.

Human prostatic cell lines.
Two prostatic cell lines BPH-1 and WPMY-1 were used for our current study.
WPMY-1, SV40 large T antigen-immortalized stromal cell line (Cat. #GNHu36, purchased from the Stem Cell Bank, Chinese Academy of Sciences, Shanghai, China), was cultured in DMEM medium (Gibco, China) containing 1% penicillin G sodium/streptomycin sulfate and 5% FBS. All cells were cultured in a humidified atmosphere consisting of 95% air and 5% CO 2 at 37 ºC. Identification of the cell lines was performed at the China Center for Type Culture Collection in Wuhan, China.

H&E Staining and Masson's Trichrome Staining.
Human prostate tissues fixed in 10% neutral buffered formalin for 48 h were processed routinely for paraffin embedding. The paraffin-embedded tissue sections (4 μm) were stained with hematoxylin and eosin using standard techniques. The paraffin sections were deparaffinized in xylene, followed by graded alcohols. and incubated with blue staining solution for 5-10 min. Next, the sections were rinsed briefly in distilled water and differentiated in 1% acetic acid solution for 2 min. After being dehydrated quickly through 95% alcohol and then absolute alcohol, the sections were cemented using neutral gum for observation. Using this procedure, prostatic SM cells were stained red, collagen fibers were stained blue and epithelial cells were stained orange. In each sample, we analyzed three areas under magnification (×100).
The choice of three fields was randomized without specific areas of a demarcated slide. The area percentage of epithelial, SM and collagen fibers were quantitated with Image Pro Plus 6.0 software, respectively.

In vitro organ bath studies.
After procurement from patients (or brain-dead men), fresh prostate specimens were immediately stored in ice-cold Krebs-Hensleit (Krebs) buffer with continuous bubbling of 95% O 2 and 5% CO 2 and then transported on ice to laboratory. Prostate specimens were cut into vertical strips in the same direction, and the dimensions of the prostate strips were approximately 1 × 0.5 × 0.5 cm. As previously described (33), human prostate strips with identical lengths were mounted longitudinally in a 10 ml During the process of equilibration, the tension of the strips was continuously adjusted to 1,000 mg. After equilibration, prostate strips were contracted with 60 mM KCl and allowed to reach the maximum force. Then the strips were washed several times with buffer and tension was brought to baseline, subsequently, cumulative concentrations (10 -7~1 0 -3 M) of phenylephrine (PE) were administered. The maximum force of KCl depolarization was taken as 100% and the force generated by PE was normalized to a percentage of this value. After the contraction experiment was completed, the strips were washed several times using buffer and the tension was reduced to the baseline. Next, strips were pre-contracted with 10 -5 M PE (a dose that induce submaximal contraction) and allowed to reach stable tension and then the relaxant effects of increasing doses of blebbistatin (BLEB) were evaluated.
Additionally, after pre-incubation with 15 mM BLEB for 30 min, its inhibitory effect on PE (10 -5 M) contractility was also tested.

Knockdown of NMMHC-A and NMMHC-B in prostatic cells.
MYH9-and MYH10-target specific small interfering RNAs (siRNAs) were
Then 10 μl CCK-8 solution was added into each well and incubated at 37°C for 1 h.
The absorbance at 450 nm was measured by a microplate reader (cat. no. SpectraMax M2; Molecular Devices, Sunnyvale, CA, USA) at the same time for each day.  Total RNA extraction and cDNA synthesis.
As described in a previous study from our group(33), total RNA was isolated from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. One μg of total RNA was reverse-transcribed to cDNA via the SuperScript II First-Strand Synthesis System according to the instructions (Invitrogen).

RT-PCR).
A total of 20 μL reaction volumes including 2 μL of the RT product cDNA Next, the PCR products were electrophoresed on a 2% agarose gel to separate

SDS-PAGE and Western-Blotting Analysis.
As previously described (34)  Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20201283

Immunohistochemistry (IHC)
As previous described (33), human prostate tissues fixed in 10% neutral buffered formalin for 48 hours were routinely processed for paraffin embedding. Samples were sectioned at 5 μm and deparaffinized in xylene followed by descending grades of ethanol (100%, 95%, 70%, 30%). Antigen retrieval was performed in 10 mM sodium citrate buffer at pH 6.0, heated to 96°C, for 30 min., followed by proteinase K treatment for 10 min. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS for 15 min. Blocking was performed by incubating sections in 5% normal donkey serum with 2% BSA for 1 hr. The sections were stained by routine IHC methods, using horse radish peroxidase polymer conjugate (Invitrogen), to localize the antibody bound to antigen, with diaminobenzidine as the final chromogen. All immunostained sections were lightly counterstained with haematoxylin. The primary antibodies (information listed in Supplementary Table 3) to target proteins were incubated for 1 hour at room temperature. Slides were evaluated for immunostaining by light microscopy.
For each slide, twenty area fields under 100 × magnification were selected randomly from each specimen (ten from stroma and another ten from epithelial compartment). For each field, IOD (integrated optical density) was calculated using Image Pro Plus 6.0 software. The mean density was calculated by IOD/area and the average values were used for target protein expression quantitative analyses.

Statistical analysis.
Results are expressed as mean ± SD for n experiments. Statistical analysis used the Student's t-test with Excel software (two-sample treatments compared). p < 0.05 was considered significant.

Histological examination for human prostate.
Human hyperplastic prostate showed obviously stroma hyperplasia accompanied with epithelial hyperplasia including epithelial layer thickening, twisted and folded, and papillary fronds protruding into the glandular cavities (Fig. 1A). In sections of Masson's trichrome staining, compared to control group, hyperplastic prostate showed increased component of epithelia (p < 0.05), SM (p < 0.05) and collagen fibers (p < 0.05) (Fig. 1B).

In vitro contractility of human hyperplastic prostate strips.
In response to adrenergic stimulation (PE), human prostate strips produced significant force in a dose-dependent manner. Notably, hyperplastic prostate strips generated more force. As shown in Fig. 2A, isolated prostatic strips from both normal human and BPH patients reached maximal contraction at 10 -4 M PE, but maximal contraction forces were around 125% and 175% of KCl depolarization induced tension, respectively.
In addition, human hyperplastic prostate exhibited a decreased shortening velocity compared to control tissue. This was reflected by a longer time to 50% PE induced maximum contraction for BPH tissue that was significantly slower than that Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20201283/900934/cs-2020-1283.pdf by guest on 05 January 2021 of controls (Fig. 2B), being 80.03±18.35 S and 48.67±10.63 S (BPH vs. control, p < 0.05), respectively. As shown in Fig. 2, the representative tracings of contraction in the response to 10 -5 M PE, hyperplastic prostate (Panel D) exhibited a slower more tonic-type contraction compared to normal prostate tissue (Panel C).

Composition of SMM II isoforms in human hyperplastic prostate is altered.
The composition of SMM II isoforms in human normal tissue (prostate, aorta, vena cava, corpus cavernosum (CC) and bladder) were detected by competitive RT-PCR (Fig. 3A). Among these tissues, aorta expressed the highest ratio of these three isoforms (90.3% SM-A, 78.3% LC 17b , 64.6% SM1), while bladder expressed the lowest (74.6% SM-A, 11.2% LC 17b , 55.3% SM1). Of note, these three isoforms in human normal prostate tissue were all between that of bladder and aorta, which was 79.9% for SM-A, 30.5% for LC 17b and 61.4% for SM1, respectively.

Blebbistatin strongly relaxes human prostate strips.
As a selective myosin II inhibitor, Blebbistatin (BLEB) relaxed PE pre-contracted human prostate SM strips in a dose-dependent manner (Fig. 4A, B). At 10 μM, BLEB could almost decrease the tension of the prostate strips to baseline. In addition, BLEB exhibited more effective relaxation on human hyperplastic prostate strips (Fig. 4C). Consistently, pre-incubation of prostate strips with 15 μM BLEB Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20201283/900934/cs-2020-1283.pdf by guest on 05 January 2021 Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20201283 effectively inhibited 10 -5 M PE-induced tension increase for both control (Fig. 4D) and BPH patients (Fig. 4E), with inhibition percentage increased by approximately 15% for prostate strips from BPH patients (Fig. 4F).

Immunohistochemistry analysis of SM MHC and NMM II in human prostate.
As shown in Figure 5, SM MHC was abundant only in the stroma component of human prostate tissue (Fig. 5A), while NMMHC-A (Fig. 5B), NMMHC-B (Fig. 5C) and NMMHC-C (Fig. 5D) were localized both in stroma and epithelial compartments.
With regard to quantitative analysis (Fig. 5E), MHC protein expression was elevated in stroma of hyperplastic prostate. Meanwhile, NMMHC-A and NMMHC-B proteins expression were also increased in both stroma and epithelial compartments of hyperplastic prostate. However, NMMHC-C protein expression showed no change between BPH and control group in either the stroma or epithelial compartments.

Expression of SM MHC and NMM II isoforms in human prostate.
The expression of SM MHC and NMM II isoforms (NMMHC-A, NMMHC-B, NMMHC-C) of human prostate were further determined by Western blotting (Fig. 6A,   B) and RT-PCR (Fig. 6C). SM MHC, NMMHC-A and NMMHC-B were upregulated in hyperplastic prostate tissue at both mRNA and protein levels. As for NMMHC-C, no alteration of expression was observed between control and BPH groups.

WPMY-1 cells inhibited prostate cell proliferation and promoted apoptosis.
To create a cell model of NMMHC-A deficiency, three distinct MYH9-target-specific-siRNAs (si-NMMHC-As) were transfected into BPH-1 and

WPMY-1 cells. After 48 h, the knockdown efficiency was validated by RT-PCR and
Western blot analysis (Fig. 7A). For both cell lines, the inhibitory efficiency of si-NMMHC-A-2 was more than 80%, therefore this siRNA was chosen for further experiment.

Discussion
Our novel data demonstrated the alteration in expression and functional activity of SMM II and NMM II isoforms in human hyperplastic prostate tissue. Human hyperplastic prostate tissue expressed more SM1 and LC 17b isoforms than normal prostate, correlating with a switch to a slower more tonic SM contraction phenotype at the functional level. In addition, the myosin II inhibitor BLEB could potently relax human prostatic SM, and it exhibited higher inhibitory efficacy for hyperplastic prostate tissue compared to normal prostate control. We also determined that NMMHC-A and NMMHC-B were upregulated in hyperplastic prostate, while knockdown of NMMHC-A or NMMHC-B could inhibit proliferation and induce apoptosis of prostatic cells.
Our recent study showed that rat prostatic SM could generate significant force in response to KCl depolarization or α1-adrenoceptor stimulus (2), which suggested this active force might play an important role in the pathophysiology of BPH. Indeed, our current study found that human prostatic SM exclusively distributed in the stroma component, which also generated significant force like rat. Moreover, we found that human hyperplastic prostate strips generated more force than normal prostate in response to PE (Fig. 2). This increase in active tension, which was mediated by the adrenergic nervous system, may contribute to the development of BPH/LUTS. Our Masson's trichrome staining revealed that the expected increase in SM content for human hyperplastic prostate (Fig. 1), which may lead to the stronger force.
Consistently, expression of SMMHC (a strong marker of the SM phenotype) was also elevated at both the mRNA and protein level in our current study (Fig. 5 & 6). In contrast, a previous study from Lin and his colleagues (35) found that SMMHC was downregulated in human hyperplastic prostate when compared with normal prostate.
However, they only detected the expression of SMMHC at the mRNA level. Moreover, previous studies demonstrated that α 1a -adrenoceptors were upregulated during BPH (36). In human prostate, α 1a -adrenoceptor is the most abundant subtype among three α 1-adrenoceptor subtypes (α 1a -, α 1b -and α 1d -), and it is expressed particularly in the prostatic stroma and mainly mediates active tension in human prostatic smooth muscle. Therefore, upregulation of this α 1-adrenoceptor subtype may also contribute to the increase in force generation. In addition, human hyperplasia prostate tissue expressed more LC 17b and SM1 isoforms than normal prostate (Fig. 3), with no change in expression ratio of SM-A, which favored a more tonic-type contraction. Previous studies had demonstrated that the relative higher ratio of LC 17b to LC 17a isoform in SM was associated with slower shortening velocity and a more tonic contraction (37)(38)(39). Indeed, we found that the time to 50% maximum contraction of prostate strips from BPH patients was longer than that of controls (in Fig. 2). Similar to the LC 17b isoform, it was demonstrated that the SM-B isoform was associated with a more phasic contraction, faster shortening velocity and higher ATPase activity, whereas the SM-A isoform was found to be expressed more in slower and more tonic type SM with lower ATPase activity (10)(11)(12).
However, the relative ratio of SM-A to SM-B was not alterations between BPH and normal groups. With regard to SM1/2 isoforms, selective knock out of the SM2 isoform (all SM1 isoform remaining) in mice was shown to lead to increases in KCl and carbachol-induced contractions (40). In addition, transgene overexpression of SM1 in mice enhanced SM contraction while transgene overexpression of SM2 attenuated SM contraction (41). Therefore, based on above two SM1/2 genetic manipulation studies, the increased expression ratio of SM1 in human hyperplastic prostatic SM (in Fig. 3) may contribute to the increased force generation which was demonstrated in our current study.
Besides the alterations in expression and functional activity of SMM II, we also observed changes in NMM II expression in hyperplastic prostate. Our recent study demonstrated that all three NMM II isoforms were richly expressed in rat prostate tissue and also expressed in human prostate cell lines (2). Consistently, our current study also identified these isoforms in human prostate tissue and additionally found NMMHC-A and NMMHC-B are upregulated in both the stroma and epithelial compartments of hyperplastic prostate (Fig. 5 & 6). Considering that NMM IIs are known to play important roles in "housekeeping" processes and are essential for tissue formation and organ development, these two upregulated isoforms (NMMHC-A and NMMHC-B) may be mechanistically associated with the occurrence and development of BPH. Indeed, our recent study found BLEB (a selective myosin ATPase inhibiting MAPK/AKT signaling pathways (43), and regulate the epithelial-mesenchymal transition (EMT) process (44)(45)(46). It is also well known that p53 is a crucial mediator of cell cycle arrest and cell apoptosis, and evidences demonstrated that accumulation of mesenchymal-like cells derived from the prostatic epithelial cells via EMT process were associated with BPH development (47)(48)(49). Interestingly, our recent microarray study also found that the MAPK pathway, which regulates cell proliferation and cell cycle, was linked to BPH (34). Therefore, these findings suggest that NMMHC-A may be involved in the development of BPH through the p53 pathway, MAPK signaling and the EMT process. In contrast, a previous study (35) showed no change in NMMHC-A expression between hyperplastic and normal prostate, which was not consistent with our current findings. However, they only examined expression at the mRNA level, while our current study performed more comprehensive studies such as RT-PCR, Western-blotting and IHC. In summary, the NMMHC-A in prostate remains less studied, and the in-depth exploration to elucidate the mechanisms involved will be intriguing. EMT process (24,50). The ECM is a complex network composed of a variety of molecules secreted by supporting cells. It not only provides an environment for cell survival and activities, but also regulates the shape, metabolism, function, migration, proliferation and differentiation of cells through signal transduction (51). Prostatic stromal-epithelial interactions are essential during development of the normal prostate(52) and associated with the etiology of BPH referred to as "reawakening" of the embryonic process (27), which suggests that NMMHC-B may be involved in the pathogenesis of BPH via regulating stromal-ECM-epithelial relationships.
NMMHC-C is a relatively newly identified NMM II isoform associated with deafness(53) and peripheral neuropathy (54). Recent studies also have demonstrated that NMMHC-C regulates cell polarity and invasion (55,56) acting as an actomyosin cytoskeleton. Its role in the prostate is unclear, however, and no expression alteration was observed between the BPH and control groups in our present study. properties, while retaining its inhibitory properties to a large extent (58)(59)(60)(61)(62). The effect of these novel BLEB derivatives on treating BPH/LUTS may be worth exploring in future.
One limitation for our current study is that the protein levels of SMM II isoforms were not determined because isoform-specific antibodies are not commercially available at present. However, a previous study demonstrated that mRNA levels of SMM II isoforms correlated well with protein expression (63).
In conclusion, we demonstrate alterations in the expression and functional activities of myosin II isoforms between hyperplastic and normal prostate, which suggests a new pathological mechanism for BPH. BLEB may be expected to be an effective potential new therapeutic for BPH/LUTS.

Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments
We thank the staff at Zhongnan Hospital of Wuhan University for their help in completing the study.     humans for each group). Data was shown as mean ± SD. *p < 0.05 vs. control; **p < 0.01 vs. control. NS means no significant differences between groups.  in stroma and epithelial compartments (n = 10 different humans for each group). The mean density was calculated by IOD/area. Data was shown as mean ± SD. * means p < 0.05 vs. control; ** means p < 0.01 vs. control.